Novel prime-boost immune-based therapy inhibiting both hepatitis B and D virus infections

Abstract

Objective: Chronic HBV/HDV infections are a major cause of liver cancer. Current treatments can only rarely eliminate HBV and HDV. Our previously developed preS1-HDAg immunotherapy could induce neutralising antibodies to HBV in vivo and raise HBV/HDV-specific T-cells. Here, we further investigate if a heterologous prime-boost strategy can circumvent T-cell tolerance and preclude HDV superinfection in vivo.

Design: A DNA prime-protein boost strategy was evaluated for immunogenicity in mice and rabbits. Its ability to circumvent T-cell tolerance was assessed in immunocompetent hepatitis B surface antigen (HBsAg)-transgenic mice. Neutralisation of HBV and HDV was evaluated both in vitro and in immunodeficient human-liver chimeric mice upon adoptive transfer.

Results: The prime-boost strategy elicits robust HBV/HDV-specific T-cells and preS1-antibodies that can effectively prevent HBV and HDV (co-)infection in vitro and in vivo. In a mouse model representing the chronic HBsAg carrier state, active immunisation primes high levels of preS1-antibodies and HDAg-specific T-cells. Moreover, transfer of vaccine-induced antibodies completely protects HBV-infected human-liver chimeric mice from HDV superinfection.

Conclusion: The herein described preS1-HDAg immunotherapy is shown to be immunogenic and vaccine-induced antibodies are highly effective at preventing HBV and HDV (super)infection both in vitro and in vivo. Our vaccine can complement current and future therapies for the control of chronic HBV and HDV infection.

Keywords: antiviral therapy; chronic viral hepatitis; hepatitis B; hepatitis D; immunotherapy.

Figure 1

Vaccine constructs and experimental overview. Four different DNA/protein preS1-HDAg fusion constructs were evaluated for immunogenicity in mice and rabbits based on a heterologous (DNA prime-protein boost) or homologous (DNA prime-boost or protein prime-boost) vaccination strategy in order to assess adaptive immune responses (T-cells and antibodies). The vaccine-induced preS1-antibodies were further evaluated for their ability to neutralise HBV and HDV both in a co-infection and superinfection setting in homozygous urokinase-type plasminogen activator-severe combined immunodeficiency (uPA^+/+-SCID) human-liver chimeric mice. Image was created with BioRender.com.

Figure 2

Induction of preS1-antibodies and HBV/HDV-specific T-cells following vaccination. C57BL/6J mice were primed with D4 DNA (50 µg) followed by in vivo electroporation and boosted twice at monthly intervals with D4 DNA, or D7 or D8 protein constructs, respectively (10 µg for each protein) or primed-boosted with D7/D8 proteins in incomplete Freud adjuvant. Two weeks after the final boost, mice were sacrificed to collect blood and splenocytes for subsequent ELISA (A) and interferon (IFN)-γ enzyme-linked immunospot assay (ELISpot) (B) analysis, respectively. (A) PreS1-antibody titres were determined as end point serum dilutions (at logarithmic scale) at which the optical density (OD) value at 405 nm was at least twice the OD of the negative control (naïve mouse serum) at the same dilution. The cut-off was set at 60, the starting dilution tested. Mean of five mice per vaccinated group is shown and each dot represents one mouse. (Bi–v) Induction of HBV/HDV-specific T-cells after 48 hours stimulation with indicated antigens. The D4 DNA prime-boost group was used as reference. Bars represent the mean number of IFN-γ spot-forming cells (SFCs) per million with SE from triplicate wells tested for each antigen condition. The different bar colours correspond to the respective sequences of the vaccine constructs, that is, green for HDAg gt1, orange for HDAg gt2 and blue for preS1A/B. HDV peptide pools 1–4 correspond to gt1 and pools 5–8 to HDAg gt2. Pools 1 and 2 of gt1 refer to sequence/isolate A, while pools 3 and 4 correspond to sequence/isolate B. Similarly for HDV gt2, pools 5 and 6 refer to sequence/isolate C and pools 7 and 8 to sequence/isolate D. Each pool contained 20 or 21 (for pools 1 and 5) 15-mer peptides with 10 amino acids (aa) overlap. PreS1A and preS1B peptides refer to the consensus sequences (total 47 aa; region 2–48 aa), while cytotoxic T-lymphocyte and T-helper ovalbumin peptides (respectively OVA CTL and OVA Th) at 10 µg/mL were used as negative controls. Concanavalin A (ConA) was used as assay positive control at 0,5 µg/mL. Image was created with BioRender.com.

Figure 3

Prevention of HBV and HDV infection in vitro. C57BL/6 mice were primed and boosted once with D7/D8 protein mix in different adjuvants (alum, MF59 and QS21). Two weeks after the last boost, serum was collected and tested for preS1 end-titres based on ELISA. The D4 DNA prime-boost group was used as reference. (A) Mean anti-preS1 end point serum dilution logarithmic scale for each vaccinated group; each symbol represents one mouse. For prevention of in vitro HBV and HDV infection, mouse sera from the preS1-HDAg protein group (D7/D8) in QS21 adjuvant was used. HepG2.hNTCP cells were incubated with a mixture of pre-incubated cell culture-derived viral inoculum and mouse preS1-HDAg antiserum (blue) or naïve mouse serum (black). Antiserum or naïve mouse serum was applied as a 1:100 dilution and 1:3 serial dilutions in culture medium. Immunofluorescence staining was performed for cells after 1 week for HBcAg (B) and HDAg (C). (B–C) HBV and HDV infection, respectively (% relative to control condition ‘infection’ which is set to an arbitrary level of 100%). (D–E) Non-linear regression modelling and IC₅₀ dilution for respectively HBV and HDV. All conditions were performed in duplicate and three images were captured per well (20×) using confocal microscopy. Infected cells were assessed using automatic cell counting (ImageJ software V.1.53c). Image was created with BioRender.com.

Figure 4

Prevention of HBV/HDV co-infection in vivo. Mouse antisera with titres ≥10⁴ (A) were passively administered through intrasplenical injection in human-liver chimeric uPA^+/+-SCID mice (hepatocyte donor C342) or with naïve serum/phosphate-buffered saline (control mice, black dots) 1 day prior to HBV/HDV co-infection (week 0) (B–C). Blue triangles (n=2): adoptive transfer of antisera obtained from D7/D8 protein mix vaccinated mice. Red triangles (n=3): adoptive transfer of antisera obtained from D4 DNA prime-D7/D8 protein mix boost group. Blood plasma was collected at week 4, 6, 8, 12, 16 and time point of sacrification (week 20). HBV DNA (B) and HDV RNA (C) levels measured in mouse plasma. Limit of detection (LOD) for HBV at 1:10 dilution is 3.750 IU/mL; LOD for HDV at 1:10 dilution is 187.5 IU/mL. PreS1A (D) and preS1B (E) antibody titres were determined in mouse plasma via ELISA as end point serum dilutions (at logarithmic scale) at which the optical density (OD) value at 405 nm was at least twice the OD of the negative control (naïve mouse serum) at the same dilution. The cut-off was set at 60.

Figure 5

Immunogenicity of heterologous prime-boost vaccination in the HBV chronic carrier. Hepatitis B surface antigens (HBsAg)-transgenic (Tg) and wild-type C57BL/6 mice were immunised with D4 DNA and boosted once (‘x1) or twice (‘x2) with D7-D8 fusion protein construct in two independent experiments. Two weeks after last boost, blood and splenocytes from each vaccinated mouse were collected for subsequent ELISA (A) and interferon (IFN)-γ enzyme-linked immunospot assay (ELISpot) (B) assays, respectively. (A) Mean anti-preS1 titres for each vaccinated group were determined as end-point serum dilutions (at logarithmic scale) at which the optical density (OD) value at 405 nm was at least twice the OD of the negative control (naïve mouse serum) at the same dilution. The cut-off was set at 60. Each symbol represents one individual mouse. (B) Number of IFN-γ secreting T-cells after 48 hours stimulation with preS1 and HDV antigens for individual HBV-Tg mice or pooled C57BL/6 mice (n=4) after one boost (x1). (Bi) Dot plots summarise the frequencies of IFN-γ-producing cells and individual dots represent the mean number of spot-forming cells per million from triplicate wells tested for each antigen condition for individual HBV-Tg mice or C57BL/6 pools. (Bii) Representative ELISpot images of IFN-γ spot-forming cells for C57BL/6 vaccinated group and HBV-Tg mice showing responses to HDV gt2C peptide pool, preS1 peptide, D7-8 fusion protein antigen and ConA. HDV peptide pools 1–4 correspond to gt1 and pools 5–8 to HDAg gt2. Pools 1 and 2 of gt1 refer to sequence/isolate A, while pools 3 and 4 correspond to sequence/isolate B. Similarly for HDV gt2, pools 5 and 6 refer to sequence/isolate C and pools 7 and 8 to sequence/isolate D. Each pool contained 20 or 21 (for pools 1 and 5) 15-mer peptides with 10 amino acids (aa) overlap. PreS1 peptide refer to the consensus sequence (total 4 7aa; region 2–48 aa), while cytotoxic T-lymphocyte and T-helper ovalbumin peptides (respectively OVA CTL and OVA Th) at 10 µg/mL were used as negative controls. Concanavalin A (ConA) was used as assay positive control at 0.5 µg/mL. Image was created with BioRender.com.

Figure 6

Prevention of HDV superinfection in vivo. (A) New Zealand white rabbits were immunised with DNA/protein preS1-HDAg harbouring regimens and induction of preS1-antibodies was assessed by ELISA. Anti-preS1 end point serum dilutions are shown at logarithmic scale. IgGs from rabbit antiserum was purified and pooled based on comparable antibody titres for use in this in vivo superinfection setting. (B–E) Six human-liver chimeric uPA^+/+-SCID mice (hepatocyte donor C342) were infected with cell-culture or patient-derived HBV at week 0 and accordingly superinfected with HDV at week 8. One day prior to superinfection, 3/6 cell-culture inoculum infected and 1/4 patient inoculum infected mice intrasplenically received 100 µL purified rabbit preS1-IgGs. In addition, these mice received repeated IgG injections (of 100 µL) intraperitoneally at day 1, 4, 7, 11 and 14 post-HDV superinfection. One mouse received the same dosing schedule, but with naïve serum, the other control mice were non-treated (black dots). Blood plasma was collected at week 4, 6, 8, 9, 10, 11, 12, 14, 16 and end point (week 17). HBV DNA (B) and HDV RNA (C) titres in mouse plasma. Limit of detection (LOD) for HBV at 1:10 dilution is 3.750 IU/ml; LOD for HDV at 1:10 dilution is 187.5 IU/mL. (D–E) PreS1A and preS1B antibody titres, respectively, represented as end point serum dilutions (at logarithmic scale) at which the optical density (OD) value at 405 nm was at least twice the OD of the negative controls at the same dilution. The cut-off is set at 60. Image was created with BioRender.com.